Interpretive Summary: In our ongoing efforts to develop countermeasures for foot-and-mouth disease virus (FMDV) infection, vaccine efficacy and potency improvement have become a primary interest. Historically, adding adjuvants to vaccine formulations greatly improves the performance of the vaccine. A new class of molecular adjuvants are molecules that stimulate the pathogen recognition receptors termed, toll like receptors (TLRs). In this manuscript, we analyzed a series of TLR stimulatory drugs that bind to TLR 7 and TLR 8. The data shows that we can use these adjuvants to stimulate cells that are critical to the immediate, nonspecific response of white blood cells, the innate response. In addition, these drugs stimulate cells that are central to the induction of long term immunity, specific for the infecting pathogen. This work provides more information for the formulation of more effective vaccines for FMDV.

Technical Abstract:
The induction of innate immune responses by toll-like receptor (TLR) agonists is the subject of intense investigation in many different species. In large part, this reflects the potential of such compounds to be effective vaccine adjuvants. For that reason, we analyzed the activation of innate cells in swine by TLR7 and TLR8 agonists. TLR7 and TLR8 agonists activated porcine NK cells by increasing IFN gamma expression and perforin storage. Activation of porcine NK cells was mediated by accessory cells, since their depletion resulted in reduced cytotoxicity towards target cells, including the human leukemia cell K562 and foot-and-mouth disease virus infected porcine cells. Accessory cells were stimulated to produce IL-12, IL-15, IL-18 and IFN alpha treatment with TLR7/8 agonists. Neutralization of these cytokines reduced but did not completely inhibit induction of NK cell cytotoxicity. Direct stimulation of NK cells with TLR7/8 agonists resulted in minimal cytotoxicity but levels of IFN gamma equivalent to those detected in the presence of accessory cells. Porcine NK cells express both TLR7 and TLR8 mRNA by real time PCR. These data indicate that TLR7 and TLR8 agonists indirectly or directly activate porcine NK cells but optimum levels of activation requires cytokine secretion by accessory cells activated by these compounds. Interestingly TLR7/8 agonists activated NK cells were cytotoxic against FMDV-infected cells in vitro indicating that these TLR agonists may be beneficial as adjuvants to stimulate the innate immunity against FMDV.